WO2019024579A1 - 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法 - Google Patents

用于检测肺癌的基因标志物、试剂盒及肺癌检测方法 Download PDF

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WO2019024579A1
WO2019024579A1 PCT/CN2018/088134 CN2018088134W WO2019024579A1 WO 2019024579 A1 WO2019024579 A1 WO 2019024579A1 CN 2018088134 W CN2018088134 W CN 2018088134W WO 2019024579 A1 WO2019024579 A1 WO 2019024579A1
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lung cancer
hydroxymethylcytosine
content
sample
gene marker
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French (fr)
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陆星宇
宋艳群
彭莱
张子谋
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上海易毕恩生物技术有限公司
上海易毕恩基因科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

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  • the invention relates to the technical field of clinical molecular diagnosis of lung cancer.
  • the present invention relates to genetic markers, kits, and lung cancer detection methods for detecting lung cancer.
  • lung cancer Primary bronchogenic lung cancer
  • IARC/WHO World Health Organization International Cancer Research Center
  • the World Health Organization International Cancer Research Center reported that the global incidence of lung cancer in men was 25.5/100,000 in 2002.
  • the Third National Survey of Death Causes of National residents released by the Ministry of Health in April 2008 shows that in the past 30 years, the incidence of lung cancer in China has increased significantly, and liver cancer has been replaced by the first cause of malignant tumor death.
  • tissue biopsy is the gold standard for clinically diagnosed lung cancer, but there are major limitations in tissue biopsy. For example, it is difficult to perform surgical sampling, or some cancer sites are inconvenient to puncture, and the puncture itself will bring certain clinical risks. Checking will bring great pain to patients.
  • the most widely used serological test is the detection of carcinoembryonic antigen (CEA), but the sensitivity and specificity of CEA for early stage lung cancer are not high.
  • the search for new markers of lung cancer is of great significance for improving the diagnosis rate of early lung cancer, achieving early intervention and reducing lung cancer mortality.
  • the present invention unexpectedly finds a plurality of highly informative information by performing high-throughput sequencing on normal samples and lung cancer samples, and analyzing the content of 5-hydroxymethylcytosine (5-hmC) in each gene. It can be used to detect genetic markers of lung cancer.
  • a first aspect of the invention relates to a genetic marker for detecting lung cancer comprising one or more of the following genes: RUNX1 transposition partner gene 1 (RUNX1T1), F-Box and leucine-rich repeats Protein 7 (FBXL7), RNCA binding motif single-chain interacting protein 3 (RBMS3), cadherin 11 (CDH11), erythrocyte membrane protein band 4.1-like protein 4A (EPB41L4A), nucleoprotein 2 (BNC2), Tolloid Protein 1 (TLL1), sulfatase 1 (SULF1), integrin subunit ⁇ 8 (ITGA8) and R-spondin 3 (RSPO3).
  • the genetic markers include RUNX1T1, FBXL7, RBMS3, CDH11, EPB41L4A, BNC2, TLL1, SULF1, ITGA8, and RSPO3.
  • the invention also relates to the use of the above gene marker in detecting lung cancer, and detecting the content of 5-hydroxymethylcytosine in the lung cancer gene marker by high-throughput sequencing, thereby determining whether the lung cancer exists.
  • a second aspect of the invention relates to a method for detecting lung cancer comprising the steps of:
  • the sample is a free DNA fragment in a subject or normal human body fluid, or is derived from intact genomic DNA in organelles, cells, and tissues.
  • body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, and the like.
  • the 5-hmC content of the genetic markers of the invention can be determined by any method known to those skilled in the art, including, for example, but not limited to, glucosylation, restriction endonucleases Method, chemical labeling method, precipitation method combined with high-throughput sequencing method, single molecule real-time sequencing method (SMRT), oxidized bisulfite sequencing method (OxBS-Seq), and the like.
  • the principle of the glucosylation method is to transfer glucose to the hydroxyl group in the presence of glucose donor substrate uridine nucleoside diphosphate glucose (UDP-Glu) using T4 phage ⁇ -glucose transferase ( ⁇ -GT).
  • ⁇ -Glucosyl-5-hydroxymethylcytosine (5-ghmC) was produced. Isotopically labeled substrates can also be used for quantification.
  • the restriction endonuclease method and the chemical labeling method were further developed on the basis of the glucosylation method. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cleavage properties of some restriction enzymes.
  • MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) And 5-hmC, but not 5-ghmC; HpaII only cleaves completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) blocks cleavage. If the CpG site contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, and there is no band in the unglycosylated control reaction; qPCR can also be used for quantitative analysis.
  • restriction enzymes also have a hindrance to 5-ghmC digestion, and can be applied to 5-hmC detection (eg, GmrSD, MspJI, PvuRts1I, TaqI, etc.).
  • the principle of the chemical labeling method is to chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, and transfer 6-N3-glucose to the position of the hydroxymethyl group to form N3-5ghmC. Subsequently, a single molecule of biotin was added to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology to analyze the distribution of 5-hmC in genomic DNA.
  • the precipitation method is to modify 5-hmC in a specific manner and then specifically capture it from the genomic DNA and perform sequencing analysis.
  • Oxidized bisulfite sequencing is the first method to quantify 5-hmC with single base resolution. Firstly, 5-hmC is subjected to KRuO4 oxidation treatment to produce 5-formylcytosine (5fC), and then heavy Sulfite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, quantitative detection of 5-hmC is performed simultaneously using a variety of detection methods.
  • the 5-hmC content of the genetic markers of the invention is determined using chemical labeling in conjunction with high throughput sequencing.
  • the method of determining the 5-hmC content of a genetic marker of the present invention comprises the steps of: fragmenting DNA from a sample of a lung cancer patient and a normal human; repairing the fragmented DNA end and The ends are filled; the end-filled DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; the DNA containing the 5-hydroxymethylcytosine tag is enriched The fragment is obtained, and the enriched product is obtained; the enriched product is subjected to PCR amplification to obtain a sequencing library; the sequencing library is subjected to high-throughput sequencing to obtain a sequencing result; and the content of 5-hydroxymethylcytosine is determined according to the sequencing result.
  • the labeling reaction comprises: i) covalent attachment of a sugar having a modifying group to a methylol group of 5-hydroxymethylcytosine using a glycosyltransferase, and ii) direct or indirect attachment of biotin Click on the chemical substrate to react with 5-hydroxymethylcytosine with a modifying group.
  • step i) and step ii) may be carried out sequentially or simultaneously in one reaction. This labeling method reduces the amount of sample required for sequencing, and the biotin tag on 5-hydroxymethylcytosine allows it to display higher kinetic signals in sequencing, improving the accuracy of nucleotide recognition.
  • the glycosyltransferase includes, but is not limited to, T4 phage ⁇ -glucosyltransferase ( ⁇ -GT), T4 bacteriophage ⁇ -glucosyltransferase ( ⁇ -GT), and the same Or a similarly active derivative, analog, or recombinase;
  • the saccharide with a modifying group includes, but is not limited to, a saccharide with an azide modification (eg, 6-N3-glucose) or with other chemical modifications a saccharide (e.g., a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, an amide group, a diene, etc.), among which a saccharide modified with azide is preferred.
  • the chemical group for indirectly linking the biotin and the click chemical substrate includes, but is not limited to, a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, Amido group, diene.
  • the DNA fragment containing the 5-hmC label is preferably enriched by a solid phase material.
  • a DNA fragment containing a 5-hydroxymethylcytosine label can be bound to a solid phase material by a solid phase affinity reaction or other specific binding reaction, and then the unbound DNA fragment can be removed by multiple washings.
  • Solid phase materials include, but are not limited to, silicon wafers or other chips with surface modification, such as artificial polymer beads (preferably 1 nm to 100 um in diameter), magnetic beads (preferably 1 nm to 100 um in diameter), agarose beads, etc. (Preferably from 1 nm to 100 um in diameter).
  • PCR amplification is preferably performed directly on the solid phase to prepare a sequencing library.
  • the amplified product can be recovered and subjected to a second round of PCR amplification to prepare a sequencing library.
  • the second round of PCR amplification can be performed using conventional methods known to those skilled in the art.
  • one or more purification steps may be further included in the process of preparing the sequencing library. Any purification kit known or commercially available to those skilled in the art can be used in the present invention. Purification methods include, but are not limited to, gel electrophoresis gel recovery, silica gel membrane spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof.
  • the sequencing library is quality checked prior to high throughput sequencing.
  • the library is subjected to fragment size analysis and the concentration of the library is absolutely quantified using the qPCR method. Sequencing libraries that pass quality checks can be used for high throughput sequencing. Then, a certain number (1-96) of libraries containing different barcodes were mixed at the same concentration and sequenced according to the standard on-line method of the second generation sequencer to obtain sequencing results.
  • Various second generation sequencing platforms and related reagents known in the art can be used in the present invention.
  • the sequencing results are preferably aligned with a standard human genome reference sequence, and the sequences in which the gene markers of the invention are aligned are selected, ie, the alignment sites and gene features (eg, groups) are selected.
  • the number of reads of the coincident region of the protein modification site, transcription factor binding site, gene exon intron region, and gene promoter, etc., to represent the level of modification of 5-hmC on the gene, thereby determining 5-hmC The amount on the genetic marker.
  • the sequencing results are first cleared of low-quality sequencing sites prior to the alignment, wherein factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences.
  • factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences.
  • determining the 5-hmC content of the gene marker means determining the 5-hmC content of the full length of the gene marker or determining the 5-hmC content of a fragment of the gene marker or combination.
  • the 5-hmC content of the corresponding gene marker in the sample of the subject is used as a reference with the 5-hmC content of the gene marker in the normal sample. standardization.
  • the 5-hmC content of the same gene marker in the normal sample and the subject sample is X and Y, respectively, and the normalized 5-hmC content of the genetic marker in the subject sample is Y/X.
  • the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, thereby obtaining a detection result based on the score.
  • “mathematical association” refers to any computational or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with the diagnosis of lung cancer.
  • different computing methods or tools can be selected to provide the mathematical associations of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, linear and Quadratic Discriminant Analysis (LQA and QDA), Naive Bayes, Random Forest, and Support Vector Machines.
  • the specific steps for mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiplying the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictor of the marker t; the predictor t of each gene marker is added to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample suffers Lung cancer; if P ⁇ 0.5, the subject sample is normal.
  • the weighting factor described herein refers to the art by the art in consideration of factors that may affect the 5-hmC content (eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.)
  • factors that may affect the 5-hmC content eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.
  • a third aspect of the present invention also relates to a kit for detecting lung cancer using the above gene marker, comprising reagents and instructions for determining a 5-hmC content of the above gene marker.
  • Agents for determining the 5-hmC content of a genetic marker are known to those skilled in the art, such as T4 bacteriophage beta-glucose transferase and isotopic labeling (for glucosylation), restriction enzymes (for restriction) Endonuclease method), glycosyltransferase and biotin (for chemical labeling), reagents for PCR and sequencing, and the like.
  • the method of detecting lung cancer of the present invention is based on the 5-hmC content of the gene marker, and thus a wider range of DNA sample sources can be used. Therefore, the present invention has the following advantages: (1) safe and non-invasive, even if the asymptomatic population has high acceptance of the test; (2) a wide range of DNA sources, no detection blind spots in imaging; (3) high accuracy It has high sensitivity and specificity for early lung cancer and is suitable for early screening of lung cancer. (4) It is easy to operate, user experience is good, and it is easy to carry out dynamic monitoring of lung cancer recurrence and metastasis.
  • the gene markers of the present invention can be combined with other clinical indicators to provide more accurate judgments for screening, diagnosis, treatment and prognosis of lung cancer.
  • Figure 1 is a graph comparing the lung cancer sample to the healthy sample control of the present invention.
  • 10 ng of plasma DNA was separately extracted from samples from 20 lung cancer patients and 20 normal humans, and this procedure can be carried out using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.
  • the amplified product was purified using Ampure XP beads to give a final sequencing library.
  • the obtained sequencing library was subjected to concentration determination by qPCR, and the DNA fragment size content in the library was determined using Agilent 2100.
  • the sequencing libraries passed the QC were mixed at the same concentration and sequenced using an Illumina Hiseq 4000.
  • the obtained sequencing results were subjected to preliminary quality control evaluation, and after the low-quality sequencing sites were cleared, the readings that met the sequencing quality standards were compared with the human standard genomic reference sequences using the Bowtie 2 tool. The number of reads was then counted using the featureCounts and HtSeq-Count tools to determine the 5-hmC content of each gene marker.
  • the factors that may affect the 5-hmC content were used as covariates, and the weighting coefficients of each gene marker were obtained by logistic regression and elastic network regularization. The results are shown in Table 1.
  • Table 1 Average normalized 5-hmC content and weighting coefficient of lung cancer gene markers of the present invention
  • the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the lung cancer sample to the average 5-hmC content of the same gene marker in the normal sample.
  • the 5-hmC content of the lung cancer gene marker of the present invention is significantly different between the normal sample and the lung cancer sample.
  • This example demonstrates the effectiveness of the lung cancer gene marker of the present invention for detecting lung cancer.
  • the 5-hmC content of the 10 lung cancer gene markers of the present invention in the first batch of 80 samples was determined according to the method of Example 1.
  • the subject sample has lung cancer; if P ⁇ 0.5, the subject sample is normal.
  • Figure 1 shows the results of distinguishing the batch of samples in accordance with the method of the present invention.
  • the method of the invention is capable of achieving a sensitivity of 89% and a specificity of 94%.

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Abstract

本发明提供了用于检测肺癌的基因标志物、试剂盒及肺癌检测方法。通过高通量测序检测RUNX1转位伴侣基因1、F-Box和富含亮氨酸重复序列的蛋白质7、RNcA结合基序单链相互作用蛋白3、钙粘着蛋白11、红细胞膜蛋白谱带4.1样蛋白4A、碱核蛋白2、Tolloid样蛋白1、硫酸酯酶1、整合素亚基α8和R-脊椎蛋白3中一个或两个以上基因中5-羟甲基胞嘧啶的含量，从而判定肺癌是否存在。

Description

用于检测肺癌的基因标志物、试剂盒及肺癌检测方法 技术领域
本发明涉及肺癌的临床分子诊断技术领域。具体的，本发明涉及用于检测肺癌的基因标志物、试剂盒及肺癌检测方法。
背景技术
原发性支气管肺癌(简称肺癌)是目前世界上最常见的恶性肿瘤之一，世界卫生组织国际癌症研究中心(IARC/WHO)报道，2002年全球肺癌男性发病率为35.5/l0万，发病97万人，死亡率为31.2/l0万，死亡85万人；女性发病率为12.1/10万，发病39万人，死亡率为10.3/10万，死亡33万人。我国卫生部2008年4月公布的《第三次全国居民死亡原因调查》显示，在过去的30年中，我国肺癌发病率增量较大，已取代肝癌成为首位恶性肿瘤死亡原因。
近年来，虽然医学技术不断发展，但肺癌患者的5年生存率并未得到明显改善，生存率仅提高到10％-15％。肺癌生存率低的一个重要原因是个体健康意识不足，对自身出现的异常现象不够重视，未及时就医，导致肺癌延迟诊断。因此，加强卫生知识宣传，大力推行个体对自身肺癌危险因素的筛查，对降低肺癌发病率、提高早期诊断率、改善患者预后有极为重要的意义。更为重要的是肺癌起病隐匿，早期常缺乏特异性表现，仅有一般的呼吸道症状，如咳嗽、痰中带血等，极易被忽略；甚至部分患者以非呼吸道症状起病，造成漏诊和误诊。 因此，大部分患者就诊时已属于晚期，五年总体生存率低于15％。但肺癌患者的预后与诊断时的临床分期密切相关，0期病人术后5年生存率可达90％以上，一期为60％，二到五期病人则从40％下降至5％以下。因此，早期诊断和早期治疗对改善肺癌患者的预后及降低死亡率有重要意义。
目前肺癌的检测主要通过影像学、组织活检及癌胚抗原(CEA)等。然而，影像学易受操作者经验影响，并且依赖于设备，费用昂贵，尤其是在医疗资源有限的情况下，其准确率难以保证，难以广泛和常规应用。组织活检是目前临床上确诊肺癌的金标准，但组织活检存在较大局限性，例如手术取样困难，或者某些癌症部位不便进行穿刺，并且穿刺本身也会带来一定的临床风险，反复穿刺筛查更会给患者带来巨大痛苦。目前应用最广的血清学检测是对癌胚抗原(CEA)的检测，但CEA对早期肺癌的灵敏度和特异性都不高。
因此，寻找新的肺癌标志物，尤其是预警监测和早期诊断的标志物对提高早期肺癌的诊断率，实现早期干预治疗，降低肺癌病死率具有非常重要的意义。
发明内容
本发明通过对正常样品和肺癌样品进行高通量测序，并对其中各基因上的5-羟甲基胞嘧啶(5-hmC)含量进行分析，出乎意料地发现了多个极具信息的可用于检测肺癌的基因标志物。
因此，本发明的第一个方面涉及用于检测肺癌的基因标志物，包括一个或两个以上以下基因：RUNX1转位伴侣基因1(RUNX1T1)、F- Box和富含亮氨酸重复序列的蛋白质7(FBXL7)、RNcA结合基序单链相互作用蛋白3(RBMS3)、钙粘着蛋白11(CDH11)、红细胞膜蛋白谱带4.1样蛋白4A(EPB41L4A)、碱核蛋白2(BNC2)、Tolloid样蛋白1(TLL1)、硫酸酯酶1(SULF1)、整合素亚基α8(ITGA8)和R-脊椎蛋白3(RSPO3)。优选的，所述基因标志物包括RUNX1T1、FBXL7、RBMS3、CDH11、EPB41L4A、BNC2、TLL1、SULF1、ITGA8和RSPO3。
本发明还涉及上述基因标志物在检测肺癌中的用途，通过高通量测序检测肺癌基因标志物中5-羟甲基胞嘧啶的含量，从而判定肺癌是否存在。
本发明的第二个方面涉及用于检测肺癌的方法，包括以下步骤：
a)测定正常样品和受试者样品中本发明所述的基因标志物的5-hmC的含量；
b)用正常样品中所述基因标志物的5-hmC含量作为参照，将受试者样品中对应的基因标志物的5-hmC含量标准化；
c)对经标准化的所述基因标志物的5-hmC含量进行数学关联，并获得评分；和
d)根据所述评分获得检测结果。
在一个实施方案中，所述样品是受试者或正常人体液中游离的DNA片段，或来源于细胞器、细胞以及组织中的完整基因组DNA。其中，体液是血液、尿液、汗液、痰液、粪便、脑脊液、腹水、胸水、胆汁、胰腺液等。
在一个实施方案中，本发明所述的基因标志物的5-hmC含量可通 过本领域技术人员已知的任何方法进行测定，例如包括但不限于，葡糖基化法、限制性内切酶法、化学标记法、与高通量测序方法联用的沉淀法、单分子实时测序法(SMRT)、氧化重亚硫酸盐测序法(OxBS-Seq)等。葡糖基化法的原理是采用T4噬菌体β-葡萄糖转移酶(β-GT),在葡萄糖供体底物尿核苷二磷酸葡萄糖(UDP-Glu)存在下,将葡萄糖转移至羟基位置,从而生成β-葡萄糖基-5-羟甲基胞嘧啶(5-ghmC)。同时可采用同位素标记底物进行定量。在葡糖基化法基础上进一步发展出限制性内切酶法和化学标记法。限制性内切酶法的原理是：葡糖基化反应改变了一些限制性内切酶的酶切特性。甲基化依赖的限制性内切酶MspI和HpaII可识别同样的序列(CCGG),但它们对甲基化状态的敏感性是不同:MspI识别并切割5-甲基胞嘧啶(5-mC)和5-hmC,但不能切割5-ghmC；HpaII只切割完全未修饰的位点,胞嘧啶上的任何修饰(5-mC、5-hmC、5-ghmC)均阻碍切割。若CpG位点含有5-hmC,那么糖基化、酶解之后能检测到条带,未糖基化对照反应中没有条带；同时可采用qPCR进行定量分析。另外,其他限制性内切酶也同样存在阻碍5-ghmC酶切的情况,可应用于5-hmC检测(如:GmrSD,MspJI,PvuRts1I,TaqI等)。化学标记法的原理是：将酶反应底物上的葡萄糖进行化学修饰转变成UDP-6-N3-glucose,将6-N3-glucose转移到羟甲基位置,生成N3-5ghmC。随后,通过点击化学方法在每个5-hmC上添加一分子生物素,结合下一代高通量DNA测序技术或单分子测序技术,可分析5-hmC在基因组DNA中的分布情况。沉淀法是将5-hmC用特殊方式修饰后再将其特异性地从基 因组DNA中捕获下来，并进行测序分析。氧化重亚硫酸盐测序法是首个以单碱基分辨率对5-hmC进行定量测序的方法.首先将5-hmC进行KRuO4氧化处理,生成5-甲酰胞嘧啶(5fC),然后采用重亚硫酸盐测序。在此过程中,5-hmC先氧化为5fC,而后脱氨形成U。通常，同时采用多种检测方法对5-hmC进行定量检测。
在本发明的一个实施方案中，利用化学标记法结合高通量测序来测定本发明的基因标志物的5-hmC含量。在该具体的实施方案中，测定本发明的基因标志物的5-hmC含量的方法包括以下步骤：将来自肺癌患者和正常人的样品的DNA片段化；将所述片段化的DNA末端修复并末端补齐；将末端补齐的DNA与测序接头连接，获得连接产物；通过标记反应对连接产物中的5-羟甲基胞嘧啶进行标记；富集含有5-羟甲基胞嘧啶标记的DNA片段，获得富集产物；对富集产物进行PCR扩增，获得测序文库；对测序文库进行高通量测序，获得测序结果；根据测序结果确定5-羟甲基胞嘧啶在基因上的含量。其中，标记反应包括：i)利用糖基转移酶将带有修饰基团的糖共价连接到5-羟甲基胞嘧啶的羟甲基上，和ii)将直接或间接连有生物素的点击化学底物与带有修饰基团的5-羟甲基胞嘧啶反应。其中，步骤i)和步骤ii)可以按顺序进行，也可以在一个反应中同时进行。这种标记方法减少了测序所需的样本量，且5-羟甲基胞嘧啶上的生物素标签使其在测序中显示出更高的动力学信号，提高了核苷酸识别的准确性。在该实施方案中，所述糖基转移酶包括但不限于：T4噬菌体β-葡糖基转移酶(β-GT)、T4噬菌体α-葡糖基转移酶(α-GT)及其具有相同 或相似活性的衍生物、类似物、或重组酶；所述带有修饰基团的糖包括但不限于：带有叠氮修饰的糖类(例如6-N3-葡萄糖)或带有其他化学修饰(例如羰基、巯基、羟基、羧基、碳-碳双键、碳-碳三键、二硫键、胺基、酰胺基、双烯等)的糖类，其中优选带有叠氮修饰的糖类；所述用于间接连接生物素和点击化学底物的化学基团包括但不限于：羰基、巯基、羟基、羧基、碳-碳双键、碳-碳三键、二硫键、胺基、酰胺基、双烯。在该实施方案中，优选通过固相材料来富集含有5-hmC标记的DNA片段。具体地，可以通过固相亲和反应或其他特异性结合反应将含有5-羟甲基胞嘧啶标记的DNA片段结合在固相材料上，然后通过多次洗涤去除未结合的DNA片段。固相材料包括但不限于带有表面修饰的硅片或其他芯片，例如人工高分子小球(优选直径为1nm-100um)、磁性小球(优选直径为1nm-100um)、琼脂糖小球等(优选直径为1nm-100um)。固相富集中所用的洗涤液是本领域技术人员熟知的缓冲液，包括但不限于：含有Tris-HCl、MOPS、HEPES(pH＝6.0-10.0，浓度在1mM到1M之间)、NaCl(0-2M)或表面活性剂如Tween20(0.01％-5％)的缓冲液。在该实施方案中，优选直接在固相上进行PCR扩增从而制备测序文库。如有需要，在固相上进行PCR扩增后，可以回收扩增产物后进行第二轮PCR扩增来制备测序文库。所述第二轮PCR扩增可用本领域技术人员已知的常规方法进行。任选地，在制备测序文库的过程中可进一步包括一个或多个纯化步骤。本领域技术人员知晓的或可商购的任何纯化试剂盒均可用于本发明。纯化方法包括但不限于：凝胶电泳切胶回收、硅胶膜离心柱法、磁珠法、乙醇或异 丙醇沉淀法或其组合。任选地，在高通量测序之前，对测序文库进行质量检查。例如，对文库进行片段大小分析并使用qPCR方法对文库的浓度进行绝对定量。通过质量检查的测序文库可用于高通量测序。然后将一定数量(1-96个)含有不同barcode的文库按相同浓度混匀并根据二代测序仪的标准上机方法上机测序，获得测序结果。本领域已知的各种二代测序平台及其相关的试剂可用于本发明。
在本发明的一个实施方案中，优选将测序结果与标准人类基因组参考序列进行比对，挑选出其中比对到本发明基因标志物上的序列，即选择比对位点与基因特征(如组蛋白修饰位点、转录因子结合位点、基因外显子内含子区域以及基因启动子等)重合区域的读段数量，以代表5-hmC在该基因上的修饰水平，从而测定5-hmC在该基因标志物上的含量。优选在进行比对前，首先将测序结果清除低质量测序位点，其中衡量测序位点质量的因素包括但不限于：碱基质量、reads质量、GC含量、重复序列和Overrepresented序列数量等。该步骤中涉及的各种比对软件和分析方法是本领域已知的。
在本发明的一个实施方案中，测定基因标志物的5-hmC含量是指测定该基因标志物全长上的5-hmC含量或测定该基因标志物上某一片段的5-hmC含量或其组合。
根据本发明，在测定各基因标志物上5-hmC含量之后，用正常样品中所述基因标志物的5-hmC含量作为参照，将受试者样品中对应的基因标志物的5-hmC含量标准化。举例而言，正常样品和受试者样品中同一基因标志物的5-hmC含量分别为X和Y，则受试者样品中该基 因标志物的标准化5-hmC含量为Y/X。
根据本发明，在数据标准化后，对各基因标志物的标准化5-hmC含量进行数学关联以获得评分，从而根据所述评分获得检测结果。如本文所用，“数学关联”是指将来自生物样品的基因标志物的5-hmC含量与肺癌诊断结果相关联的任何计算方法或机器学习方法。本领域普通技术人员理解，可选择不同的计算方法或工具用于提供本发明的数学关联，例如弹性网络正则化、决策树、广义线性模型、逻辑回归、最高分值对、神经网络、线性和二次判别式分析(LQA和QDA)、朴素贝叶斯、随机森林和支持向量机。
在本发明的一个实施方案中，对各基因标志物的标准化5-hmC含量进行数学关联并获得评分的具体步骤如下：将各基因标志物的标准化5-hmC含量乘以加权系数，获得该基因标志物的预测因子t；将各基因标志物的预测因子t相加，获得总预测因子T；将总预测因子T经过Logistic转换获得评分P；若P>0.5，则该受试者样品患有肺癌；若P≤0.5，则该受试者样品为正常。本文所述的加权系数是指在考虑可能影响5-hmC含量的因素(例如受试者地域、年龄、性别、低于、吸烟史、饮酒史、家族史等)的情况下，通过本领域技术人员已知的各种高级统计分析方法获得的系数。
本发明第三个方面还涉及利用上述基因标志物进行肺癌检测的试剂盒，其包括用于测定上述基因标志物的5-hmC含量的试剂和说明书。用于测定基因标志物的5-hmC含量的试剂是本领域技术人员已知的，例如T4噬菌体β-葡萄糖转移酶和同位素标记(对于葡糖基化 法)、限制性内切酶(对于限制性内切酶法)、糖基转移酶和生物素(对于化学标记法)、PCR和测序所用试剂等。
与现有技术相比，本发明检测肺癌的方法是基于基因标志物上的5-hmC含量，因此可以使用更为广泛的DNA样品来源。因此，本发明具有以下几个优点：(1)安全无创，即使无症状人群也对该检测接受度高；(2)DNA来源广泛，不存在影像学中的检测盲区；(3)准确性高，对早期肺癌有较高的灵敏度和特异性，适合用于肺癌的早期筛查；(4)操作方便，用户体验好，容易进行肺癌复发和转移的动态监测。本发明的基因标志物可与其他临床指标相结合，为肺癌筛查、诊断、治疗与预后提供更准确的判断。
附图说明
图1是本发明区分肺癌样品与健康样品对照的曲线图。
具体实施方式
下面结合实施例及附图对本发明进行详细说明，以使本领域技术人员更好的理解本发明，并能予以实施。
实施例1.肺癌基因标志物的筛选
1)抽提血浆DNA：
从来自20位肺癌患者和20位正常人的样品中分别抽提10ng血浆DNA，可利用本领域技术人员所熟知的任何适用于抽提血浆DNA的方法和试剂进行此步骤。
2)将血浆DNA进行末端补齐、悬A并与测序接头连接：
根据Kapa Hyper Perp Kit说明书制备含有50uL血浆DNA、7uL  End Repair & A-Tailing Buffer和3uL End Repair & A-Tailing Enzyme mix的反应混合液(总体积为60uL)，在20℃温浴30分钟，然后在65℃温浴30分钟。在1.5mL低吸附EP管中配置以下连接反应混合物：5uL Nuclease free water，30uL Ligation Buffer以及10uL DNA Ligase。向45uL连接反应混合物中加入5uL的测序接头，混合，于20℃加热20分钟，然后保持于4℃。使用AmpureXP beads对反应产物进行纯化，用20uL含Tris-HCl(10mM，pH＝8.0)及EDTA(0.1mM)的缓冲液进行洗脱获得最终的DNA连接样品。
3)标记5-羟甲基胞嘧啶：
制备总体积为26uL的标记反应混合液：叠氮修饰的二磷酸尿苷葡萄糖(即UDP-N3-Glu，终浓度为50uM)、β-GT(终浓度为1uM)、Mg2+(终浓度为25mM)、HEPES(pH＝8.0，终浓度为50mM)和来自上述步骤的20uL DNA。将混合液在37℃温浴1小时。取出混合液，用AmpureXP beads纯化，获得纯化的20uL DNA。
然后在上述纯化的20uL DNA中加入1uL连接有生物素的二苯基环辛炔(DBCO-Biotin)，于37℃反应2小时，接着用AmpureXP beads纯化，获得纯化的标记产物。
4)固相富集含有标记的5-羟甲基胞嘧啶的DNA片段：
首先，按以下步骤准备磁珠：取出0.5uL C1 streptadvin beads(life technology)并加入100uL缓冲液(5mM Tris，pH＝7.5，1M NaCl，0.02％Tween20)，涡旋混合30秒，然后用100uL洗涤液(5mM Tris，pH＝7.5，1M NaCl，0.02％Tween20)洗涤磁珠3次，最后加入 25uL结合缓冲液(10mM Tris，pH＝7.5，2M NaCl，0.04％Tween20或其他表面活性剂)，并混合均匀。然后，在磁珠混合液中加入上述步骤获得的纯化的标记产物，并在旋转混合器中混合15min使其充分结合。最后，用100uL洗涤液(5mM Tris，pH＝7.5，1M NaCl，0.02％Tween20)洗涤磁珠3次，离心去掉上清液，加入23.75uL不含核酸酶的水。
5)PCR扩增：
向上述步骤的最终体系中加入25uL的2X PCR master mix和1.25uL PCR引物(总体积为50uL)，按照下述PCR反应循环的温度和条件进行扩增：
Figure PCTCN2018088134-appb-000001
将扩增产物用AmpureXP beads纯化，得到最终测序文库。
6)对测序文库进行质检后进行高通量测序：
将获得的测序文库通过qPCR进行浓度测定，并用Agilent2100对文库中DNA片段大小含量进行确定。将通过质检的测序文库以相同浓度混合，用Illumina Hiseq 4000进行测序。
7)确定各基因标志物的5-hmC含量和加权系数：
将获得的测序结果进行初步质控评估，清除低质量测序位点后，将达到测序质量标准的读段利用Bowtie2工具与人类标准基因组参考序列进行比较。然后利用featureCounts和HtSeq-Count工具来统 计读段数量以确定各基因标志物的5-hmC含量。同时利用高通量测序结果，将可能影响5-hmC含量的因素作为共变量，通过逻辑回归和弹性网络正则化获得各基因标志物的加权系数。结果如表1所示。
表1：本发明的肺癌基因标志物的平均标准化5-hmC含量和加权系数
Figure PCTCN2018088134-appb-000002
如上所述，平均标准化5-hmC含量是指肺癌样品中该基因标志物的平均5-hmC含量与正常样品中同一基因标志物的平均5-hmC含量之比。从表1可以看出，本发明的肺癌基因标志物的5-hmC含量在正常样品中和肺癌样品中存在显著差异。
实施例2.肺癌基因标志物的有效性
本实施例验证本发明的肺癌基因标志物用于检测肺癌的有效性。
根据实施例1的方法测定第一批80个样品(40例肺癌和40例健康对照)中本发明所述的10个肺癌基因标志物的5-hmC含量。
将各基因标志物的标准化5-hmC含量乘以该标志物在实施例1中对应的加权系数，获得该基因标志物的预测因子t，之后将各基因标 志物的预测因子t相加，获得总预测因子T，然后将总预测因子T根据以下公式经过Logistic转换获得评分P：
Figure PCTCN2018088134-appb-000003
若P>0.5，则该受试者样品患有肺癌；若P≤0.5，则该受试者样品为正常。
图1示出了根据本发明的方法区分该批样品的结果。如图1所示，本发明的方法能够达到89％的灵敏度和94％的特异性。
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 基因标志物用于检测肺癌的用途，通过高通量测序检测肺癌基因标志物中5-羟甲基胞嘧啶的含量，从而判定肺癌是否存在，所述基因标志物包括一个或两个以上以下基因：RUNX1转位伴侣基因1(RUNX1T1)、F-Box和富含亮氨酸重复序列的蛋白质7(FBXL7)、RNcA结合基序单链相互作用蛋白3(RBMS3)、钙粘着蛋白11(CDH11)、红细胞膜蛋白谱带4.1样蛋白4A(EPB41L4A)、碱核蛋白2(BNC2)、Tolloid样蛋白1(TLL1)、硫酸酯酶1(SULF1)、整合素亚基α8(ITGA8)和R-脊椎蛋白3(RSPO3)。
  2. 根据权利要求1所述的用途，其特征在于：所述基因标志物包括RUNX1T1、FBXL7、RBMS3、CDH11、EPB41L4A、BNC2、TLL1、SULF1、ITGA8和RSPO3。
  3. 一种用于检测肺癌的方法，其特征在于包括以下步骤：
    a)测定正常样品和受试者样品中权利要求1和2所述的基因标志物的5-羟甲基胞嘧啶的含量；
    b)用正常样品中所述基因标志物的5-羟甲基胞嘧啶含量作为参照，将受试者样品中对应的基因标志物的5-羟甲基胞嘧啶含量标准化，正常样品和受试者样品中同一基因标志物的5-羟甲基胞嘧啶含量分别为X和Y，则受试者样品中该基因标志物的标准化5-羟甲基胞嘧啶含量为Y/X；
    c)对步骤b)中经标准化的所述基因标志物的5-羟甲基胞嘧啶含量进行数学关联，并获得评分P；
    d)根据所述评分P的数值大小得到受试者样品是否患有肺癌的检测结果。
  4. 根据权利要求3所述的方法，其特征在于步骤a)包括如下步骤：将来自肺癌患者和正常人的样品的DNA片段化；将所述片段化的DNA末端修复并末端补齐；将末端补齐的DNA与测序接头连接，获得连接产物；通过标记反应对连接产物中的5-羟甲基胞嘧啶进行标记；富集含有5-羟甲基胞嘧啶标记的DNA片段，获得富集产物；对富集产物进行PCR扩增，获得测序文库；对测序文库进行高通量测序，获得测序结果；根据测序结果确定5-羟甲基胞嘧啶在基因上的含量。
  5. 根据权利要求4所述的方法，其特征在于：所述标记反应包括：i)利用糖基转移酶将带有修饰基团的糖共价连接到5-羟甲基胞嘧啶的羟甲基上，和ii)将直接或间接连有生物素的点击化学底物与带有修饰基团的5-羟甲基胞嘧啶反应。
  6. 根据权利要求3、4或5所述的方法，其特征在于：所述步骤(a)是测定所述基因标志物全长或其片段上的5-羟甲基胞嘧啶的含量。
  7. 根据权利要求3、4或5所述的方法，其特征在于步骤c)包括如下步骤：将各基因标志物的标准化5-羟甲基胞嘧啶含量乘以加权系数，获得该基因标志物的预测因子t；将各基因标志物的预测因子t相加，获得总预测因子T；将总预测因子T经过Logistic转换获得评分P；若P>0.5，则该受试者样品患有肺癌；若P≤0.5，则该受试者样品为正常。
  8. 根据权利要求3所述的方法，其特征在于：所述样品是来自正常人或受试者体液中游离的DNA片段，或来源于细胞器、细胞以及组织中的完整基因组DNA。
  9. 根据权利要求8所述的方法，其特征在于：所述体液是血液、尿液、汗液、痰液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液。
  10. 一种用于检测肺癌的试剂盒，其特征在于包括：
    a)用于测定权利要求1所述基因标志物的5-羟甲基胞嘧啶含量的试剂；和b)说明书；
    所述5-羟甲基胞嘧啶含量是指所述基因标志物全长或其片段上的5-羟甲基胞嘧啶的含量。
PCT/CN2018/088134 2017-08-04 2018-05-24 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法 WO2019024579A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN107365845A (zh) * 2017-08-04 2017-11-21 上海易毕恩生物技术有限公司 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法
CN107385051A (zh) * 2017-08-04 2017-11-24 上海易毕恩生物技术有限公司 用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法
CN108103196A (zh) * 2018-02-05 2018-06-01 上海易毕恩生物技术有限公司 一种用于检测食管癌的基因标志物及其用途和检测方法
CN112129938B (zh) * 2019-06-25 2023-08-18 中国科学院分子细胞科学卓越创新中心 UDP-Glc在肺癌转移评估中的应用
CN112175953B (zh) * 2020-03-13 2022-05-24 芯超生物科技(河南)有限公司 一种基因抑制剂在制备肺癌药物中的应用
CN113430255A (zh) * 2021-07-19 2021-09-24 深圳泰莱生物科技有限公司 基于5hmC点击化学高通量测序技术的肺癌检测方法
CN113584168A (zh) * 2021-07-19 2021-11-02 深圳泰莱生物科技有限公司 基于甲基化免疫沉淀高通量测序技术的肺癌检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091492A (zh) * 2011-11-04 2013-05-08 中国科学院上海生命科学研究院 诊断癌症的试剂及试剂盒
CN106755464A (zh) * 2017-01-11 2017-05-31 上海易毕恩基因科技有限公司 用于筛选肠癌和/或胃癌的基因标志物的方法、用该方法筛选的基因标志物及其用途
CN107142320A (zh) * 2017-06-16 2017-09-08 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107164508A (zh) * 2017-06-16 2017-09-15 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107365845A (zh) * 2017-08-04 2017-11-21 上海易毕恩生物技术有限公司 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法
CN107385050A (zh) * 2017-08-04 2017-11-24 上海易毕恩生物技术有限公司 用于检测胰腺癌的基因标志物、试剂盒及胰腺癌检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140030727A1 (en) * 2012-01-20 2014-01-30 Gerd PFEIFER Loss of 5-hydroxymethylcytosine as a biomarker for cancer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091492A (zh) * 2011-11-04 2013-05-08 中国科学院上海生命科学研究院 诊断癌症的试剂及试剂盒
CN106755464A (zh) * 2017-01-11 2017-05-31 上海易毕恩基因科技有限公司 用于筛选肠癌和/或胃癌的基因标志物的方法、用该方法筛选的基因标志物及其用途
CN107142320A (zh) * 2017-06-16 2017-09-08 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107164508A (zh) * 2017-06-16 2017-09-15 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107365845A (zh) * 2017-08-04 2017-11-21 上海易毕恩生物技术有限公司 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法
CN107385050A (zh) * 2017-08-04 2017-11-24 上海易毕恩生物技术有限公司 用于检测胰腺癌的基因标志物、试剂盒及胰腺癌检测方法

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